PUMA is a BH3-only protein that elicits the initiation of apoptosis in response to genotoxic and non-genotoxic stresses. PUMA binds to members of the anti-apoptotic Bcl-2 family of proteins, such as Bcl-2, Bcl-XL, and Mcl-1, to release Bax and trigger a multitude of downstream events leading to apoptosis. PUMA and apoptosis induction by genotoxic stress is dependent on p53, a tumor suppressor inactivated in ~50% of cancers. Recently, our laboratory discovered that PUMA is necessary for apoptosis induced by non-genotoxic agents, such as the kinase inhibitor staurosporine and its analogue UCN-01, through p53-independent mechanisms. Preliminary studies suggest that the transcription factor forkhead box O3A (FoxO3a) is responsible for PUMA activation by kinase inhibitors. I propose to test the hypothesis that FoxO3a-mediated PUMA induction can be used to restore apoptosis regulation in p53-deficient human cancer cells. Aim 1 will investigate the mechanism of p53-independent induction of PUMA by kinase inhibitors in colorectal cancer cells. I will use siRNA, western blotting, luciferase assays, and chromatin immunoprecipitation to determine whether FoxO3a plays a direct role in PUMA induction following kinase inhibitor treatment. I will also explore the upstream pathways through which kinase inhibition activates FoxO3a and PUMA. Aim 2 will determine the functional role of PUMA in apoptosis induced by kinase inhibitors and its therapeutic effect in vivo. I will determine if PUMA is required for the apoptotic events induced by the kinase inhibitor UCN-01, and if PUMA is required for the chemosensitization effect of UCN- 01. I will also investigate if PUMA mediates the anti-tumor effect of UCN-01 in vivo by promoting apoptosis. Aim 3 is designed to develop a screening assay for identifying novel agents that activate PUMA in p53-deficient human cancer cells. I will engineer a stable p53-deficient cell line expressing a luciferase reporter driven by the FoxO3a response elements from the PUMA promoter. In collaboration with my Co-Sponsor's laboratory, I will use this cell line to screen FDA-approved drugs via a high-throughput assay to identify agents that induce PUMA independently of p53. The positive agents will be further tested for their ability to induce PUMA and apoptosis in other colorectal cancer cell lines. PUBLIC HEALTH RELEVANCE: Selective killing of cancer cells is essential for anti-cancer therapies. Restoring apoptosis in human cancer cells by activating p53-independent PUMA induction pathways may afford improved therapeutic strategies and agents.